Isolation and purification of total RNA from Streptococcus mutans in suspension cultures and biofilms.
The presence of extracellular polysaccharides matrix makes extraction and purification of RNA from Streptococcus mutans within biofilms challenging. In this study, several approaches to purify RNA extracted from S. mutans in suspension cultures and biofilms were examined. The combination of sonication (3 pulses of 30 s at 7 W), suspension in NAES buffer (50 mM sodium acetate buffer, 10 mM EDTA and 1% SDS; pH 5.0) and homogenization-mechanical cells disruption in NAES- acid phenol:chloroform, yielded 9.04 mg (or 0.52 mg) of crude preparation of RNA per 100 mg of total cell (or biofilm) dry-weight. The crude RNA preparations were subjected to various DNAse I treatments. The combination of DNAse I in silica-gel based column followed by recombinant DNase I in solution provided the best genomic DNA removal, resulting in 4.35 mg (or 0.06 mg) of purified RNA per 100 mg of total cell (or biofilm) dry-weight. The cDNAs generated from the purified RNA sample were efficiently amplified using gtfB S. mutans-specific primers. The results showed a method that yields high-quality RNA from both planktonic cells and biofilms of S. mutans in sufficient quantity and quality for real-time RT-PCR analyses.
Cury
Jaime Aparecido
JA
Piracicaba Dental School, State University of Campinas, Piracicaba, SP, Brazil.
Seils
Jennifer
J
Koo
Hyun
H
D018441:Biofilms; D002478:Cells, Cultured; D010933:Plankton / Q000737:chemistry*; D011134:Polysaccharides / Q000096:biosynthesis / Q000737:chemistry*; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013295:Streptococcus mutans / Q000235:genetics*
D011134:Polysaccharides; D012329:RNA, Bacterial